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- Table of Contents
SPIB / Transcription factor Spi-B · Western blot design guide
Design a Western Blot for SPIB
Source-linked SPIB Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SPIB WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
SPIB Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~28.8 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Spleen (IHC candidate; verify WB) +4 more | |
| Negative control | Esophagus (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked SPIB Western Blot Protocol Options
The A02805-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human prostate tissue lysate (catalog A02805-1) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02805-1 · 1 μg/ml (catalog A02805-1) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected SPIB Western Blot Band Size?
SPIB is predicted at 28.8 kDa; its isoforms could affect migration, but no empirical band size or distinct isoform pattern is supplied.
| Band near 28.8 kDa | Consistent with the predicted SPIB mass; confirm identity with a specificity control. |
| Several discrete bands | Could reflect isoforms 1, 2, and 3; their migration differences are unknown. |
| One band despite three isoforms | The isoforms may not resolve or may not all be expressed in this sample. |
| Stronger band in nuclear extract than whole-cell lysate | Consistent with the reported nuclear location of isoform 1. |
💡Expected SPIB appearanceSPIB has a predicted mass of 28.8 kDa; no empirical band size is supplied, and the three isoforms have no established migration pattern, so confirm any candidate band with a specificity control.
| UniProt predicted mass | Provides a 28.8 kDa reference for SPIB. |
| Isoform 1 | Its apparent size relative to the other isoforms is not supplied. |
| Isoform 2 | Alternative splicing may change its size, but its migration is unknown. |
| Isoform 3 | Alternative splicing may change its size, but its migration is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear isoform 1 may be difficult to detect in whole-cell lysate. | Check a nuclear extract and an appropriate positive control. |
| Band higher than expected | An isoform is possible, but isoform sizes are unknown. | Check band identity with SPIB depletion or an independent antibody. |
| Band lower than expected | An isoform is possible, but no cleavage feature or isoform mass is supplied. | Check band identity with SPIB depletion and assess sample integrity. |
| Multiple bands | SPIB has three named isoforms, though distinct bands are not established. | Compare bands after SPIB depletion or with an independent antibody. |
| Weak or no signal | Nuclear localization of isoform 1 may limit its signal in a whole-cell sample. | Test a nuclear-enriched sample alongside a positive control. |
Experiment evidence
Western blot images showing SPIB band
A02805-1 · SPIB
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SPIB Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SPIB in Western blot, you can use spleen tissue, where HPA reports high expression.
Positive control: Spleen (IHC candidate; verify WB)
Negative control: Esophagus (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Because SPIB is nuclear, nuclear extracts may improve detection.
HPA tissue expression evidence for SPIB
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Spleen | cells in red pulp | High | Protein (IHC) | HPA → |
| Tonsil | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SPIB Western Blot Tips
Deeper troubleshooting and optimisation questions for SPIB, answered from its protein features.
How should SPIB band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could SPIB isoforms produce bands at different sizes?
Isoforms · Yes. UniProt lists three isoforms. Isoform 2 replaces residues 163–177 and lacks residues 178–262; isoform 3 lacks residues 1–91 and replaces residues 92–113. Both differ in length from the canonical 262-residue isoform, but these features do not establish their apparent band positions.
Check its epitope against the listed sequence changes. An epitope within residues 178–262 would be absent from isoform 2; one within residues 1–91 would be absent from isoform 3. The replacement regions at 163–177 and 92–113 may also affect recognition. Confirm the antibody’s stated epitope before comparing isoforms.
A lower band alone cannot identify isoform 2. Its sequence lacks canonical residues 178–262 and replaces residues 163–177, but no observed band positions are supplied. An antibody recognizing the missing region would not detect it; an isoform-specific reagent or sequence-based confirmation would strengthen the assignment.
Check its epitope against the listed sequence changes. An epitope within residues 178–262 would be absent from isoform 2; one within residues 1–91 would be absent from isoform 3. The replacement regions at 163–177 and 92–113 may also affect recognition. Confirm the antibody’s stated epitope before comparing isoforms.
A lower band alone cannot identify isoform 2. Its sequence lacks canonical residues 178–262 and replaces residues 163–177, but no observed band positions are supplied. An antibody recognizing the missing region would not detect it; an isoform-specific reagent or sequence-based confirmation would strengthen the assignment.
Do SPIB features support a modification-induced band shift?
PTM · No modified residues or glycosylation sites are listed in the supplied features. A shifted band cannot be attributed to a specific modification from this record. Check the three alternative sequences and antibody epitope when interpreting it; feature presence or absence alone does not establish why a band migrates differently.
Does this guide establish induction of SPIB?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
How should transfer be checked for SPIB?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02805-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should I quantify multiple SPIB-reactive bands?
Quantitation · First determine which bands the antibody can recognize from its epitope and the three isoform sequences. Quantify a defined band consistently across samples, or report bands separately if their identities are uncertain. The features provide no observed band positions, so do not assign an isoform solely by apparent mass.
How should I interpret a band away from SPIB’s predicted mass?
Interpretation · The 262-residue canonical sequence has a predicted mass of 28.8 kDa. No observed band position is supplied, so a difference cannot be assigned a cause from these features alone. Compare the band with a molecular-weight marker and assess whether the antibody could detect an alternative isoform.
UniProt places isoform 1 in the nucleus and identifies SPIB as a DNA-binding transcription factor. Nuclear samples are therefore relevant for measuring isoform 1. Keep sample preparation consistent across comparisons; the supplied features do not establish the localization of isoforms 2 or 3.
UniProt places isoform 1 in the nucleus and identifies SPIB as a DNA-binding transcription factor. Nuclear samples are therefore relevant for measuring isoform 1. Keep sample preparation consistent across comparisons; the supplied features do not establish the localization of isoforms 2 or 3.
Boster reagents
SPIB Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-Transcription factor Spi-B SPIB Antibody
Cat # A02805-1
The catalog reports one anti-SPIB antibody, A02805-1, with reported Human, Mouse, and Rat reactivity. Its Western blot image uses human prostate tissue lysate at 1 μg/ml; the supplied evidence does not show Mouse or Rat blots.
Which to pick: A02805-1 is the only listed option and has a Western blot image from human prostate tissue lysate. For Mouse or Rat samples, reactivity is listed, but no corresponding Western blot image is supplied.
Source: BosterBio SPIB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q01892.
- Human Protein Atlas. SPIB tissue expression.
- PMC8044826 — target-verified WB comparison
- PMC12998320 — target-verified WB comparison